The oligosaccharide moieties of glycoconjugates play important roles in several biological processes of a cell, including the folding and transport of glycoproteins across cellular compartments. For the biosynthesis of these complex oligosaccharides, an intricate machinery exists in a cell. Defective glycan synthesis has serious pathological consequences and results in several human diseases. The oligosaccharide moieties bind to cellular proteins with high specificity and modulate the homo- and hetrodimerization of glycoproteins. Due to the conformational flexibility of oligosaccharides, the torsional angles of a disaccharide unit, especially around the a1-6-linkage, adjust in such a way that the side groups of the oligosaccharides orient themselves in a manner that promotes favorable interactions with the binding residues of the protein. Branched oligosaccharides cross-link proteins and generate infinite networks of protein-carbohydrate complexes, resulting in the modulation of various cell responses. Defining the oligosaccharide binding site of Gal-T1 by docking oligosaccharides into the binding site and MD simulation of the complexes: We have continued to use molecular modeling methods to study the binding of oligosaccharides to proteins, in particular the binding of various oligosaccharide substrates to Gal-T1, the three dimensional structure of which has recently been determined in our laboratory, either in complex with UDP-galactose and Mn2+ion, or in complex with a-lactalbumin and N-acetylglucosamine (see Project # Z01 BC 009305-06 LECB). Examination of the GlcNAc binding site in Gal-T1 from the Gal-T1 LA GlcNAc crystal structure reveals an "open canal shaped" extended sugar binding site that lies behind the GlcNAc binding site, with an average width and length of 10 ? and 16 ?, respectively. This site is formed by the residues from three regions; residues 280 to 289, residues 319 to 325, and residues 359 to 368. LA binds to this region in the crystal structure of Gal-T1-LA complex; therefore, it is expected to compete with the GlcNAc containing oligosaccharides such as chitobiose. A limited number of preferred oligosaccharides as substrates for Gal-T1 are known. These studies have shown that among the different GlcNAc containing disaccharides, only a b-linked disaccharide such as GlcNAcb1,4-GlcNAc or GlcNAcb1,2-Man is preferred over a-linked disaccharides. In fact a-methyl-GlcNAc is less preferred compared to GlcNAc by itself. Also, oligosaccharides such as N-glycans are more preferred acceptor substrates than a (GlcNAc)4. The presence of the protein or peptide attached to such an N-glycan does not influence the binding of an oligosaccharide. In order to probe the size and nature of the oligosaccharide binding site, a modeling study of the docking of various disaccharides and N-glycan ligands in the binding site were carried out. Each ligand conformation, inter-molecular interaction energy between Gal-T1 and the saccharide was calculated using CVFF force field and Discover module of InsightII. Only protein residues within 9 A from any of the ligand atoms were considered for energy calculations. The conformation of the ligand was varied in the binding site by systematically scanning the entire stereochemically allowed region of the ligand. The total energy, comprising the intermolecular protein-ligand interaction energy and intermolecular ligand energy, was used as a guide in determining the possible allowed conformations of the ligand in the binding site. These modeling studies show that GlcNAc with an a-linked substitution such as a-benzyl-GlcNAc can not bind to Gal-T1 because of severe steric contacts with the highly conserved Tyr286 residue, whereas GlcNAc with a b-linked substitution such as b-benzyl-GlcNAc can bind without any steric contacts. Docking of a biantennary N-glycan with GlcNAcs at its reducing ends in the extended sugar binding site reveals that the acceptor binding site in Gal-T1 can accommodate a linear pentasaccharide all the way from the GlcNAc moiety to the aspargine-linked GlcNAc. The binding site can also accommodate either the a-1-3 arm (GlcNAc3b1-2Man3a1-3Manmb1-4GlcNAcb1-4GlcNAc-N) or a-1-6 arm (GlcNAc6b1-2Man6a1-6Manmb1-4GlcNAcb 1-4GlcNAc-N) of the N-glycan without any steric hindrance. MD simulations in water (75 ? cube) for 750 ps of Gal-T1 alone or with the docked bi-antennary oligosaccharide in the binding site show that the conformational changes in the y at the Mana1-3Manm and GlcNAc6b1-2Man6 linkage are pronounced in the bound 1-3-arm of the bi-antennary oligosaccharide. Conformational changes in y at the Manmb1-4GlcNAc linkage, and f at the Man6a1-6Manm linkage are pronounced in the bound 1-6-arm of the bi-antennary oligosaccharide. The MD simulation results suggest hydrophobic interactions between the oligosaccharide and Gal-T1 when 1-3-arm is bound in the binding site, thus favoring the transfer of galactose to the GlcNAc on the 1-3-arm of the bi-antennary oligosaccharide. This conclusion concurs with the published biochemical data. In humans Gal-T1 family members are responsible for the synthesis of Gal moiety in different oligosaccharides, indicating that although all these enzymes transfer Gal to GlcNAc, each recognizes the remaining oligosaccharide moieties to which GlcNAc is attached differently. The sequence comparison of the human Gal-T family members reveals only a little or no variation in the GlcNAc binding site among the family members where as the extended oligosaccharide binding region shows significant variations, indicating that these enzymes may prefer different GlcNAc-containing oligosaccharides as their preferred sugar acceptors. To determine the exact mode of binding of the oligosaccharide in the binding site, as a first attempt we have been successful in co-crystallizing Gal-T1 mutants with di and a penta-saccharide. The crystal structure of these complexes is currently being determined.